Analysis of area-specific expression patterns of RORbeta, ER81 and Nurr1 mRNAs in rat neocortex by double in situ hybridization and cortical box method.

Hirokawa J, Watakabe A, Ohsawa S, Yamamori T - PLoS ONE (2008)

Bottom Line:
Second, at single cell level, RORbeta and ER81 mRNAs were coexpressed in a subpopulation of layer 5 neurons, whereas Nurr1 and ER81 mRNAs were not colocalized.The tight relationship between the expression of the three layer specific genes and functional areas were revealed, demonstrating the usefulness of cortical box method in the study on the cerebral cortex.In particular, it allowed us to perform statistical evaluation and pattern matching, which would become important in interpreting the ever-increasing data of gene expression in the cortex.

Background: The mammalian neocortex is subdivided into many areas, each of which exhibits distinctive lamina architecture. To investigate such area differences in detail, we chose three genes for comparative analyses, namely, RORbeta, ER81 and Nurr1, mRNAs of which have been reported to be mainly expressed in layers 4, 5 and 6, respectively. To analyze their qualitative and quantitative coexpression profiles in the rat neocortex, we used double in situ hybridization (ISH) histochemistry and cortical box method which we previously developed to integrate the data of different staining and individuals in a standard three-dimensional space.

Principal findings: Our new approach resulted in three main observations. First, the three genes showed unique area distribution patterns that are mostly complementary to one another. The patterns revealed by cortical box method matched well with the cytoarchitectonic areas defined by Nissl staining. Second, at single cell level, RORbeta and ER81 mRNAs were coexpressed in a subpopulation of layer 5 neurons, whereas Nurr1 and ER81 mRNAs were not colocalized. Third, principal component analysis showed that the order of hierarchical processing in the cortex correlates well with the expression profiles of these three genes. Based on this analysis, the dysgranular zone (DZ) in the somatosensory area was considered to exhibit a profile of a higher order area, which is consistent with previous proposal.

Conclusions/significance: The tight relationship between the expression of the three layer specific genes and functional areas were revealed, demonstrating the usefulness of cortical box method in the study on the cerebral cortex. In particular, it allowed us to perform statistical evaluation and pattern matching, which would become important in interpreting the ever-increasing data of gene expression in the cortex.

pone-0003266-g001: Double in situ hybridization histochemistry (ISH) of RORbeta/ER81 (A) and RORbeta/Nurr1 (B).Signals in red are for RORbeta and those in green are for ER81 (A) or Nurr1 (B). The arrowheads indicate the area borders that were deduced by comparing the gene expression patterns shown by double ISH and those revealed by the cortical box method. Par1, Par2, Oc1, Oc2L and Te1 correspond to the primary and secondary somatosensory areas (Par1 and Par2), the primary and secondary visual areas (Oc1 and Oc2L) and the primary auditory area (Te1). Ectorhinal cortex (Ect) is also indicated. The white bars denoted as a–e and a'–e' are the regions magnified in Fig. 2. This figure is a montage of several images. Although the lighting condition of the original images was not even at this low resolution, we adjusted the contrast of each component image manually so that the montage appeared to be consecutive. Scale bar, 2 mm.

Mentions:
Figure 1A shows the double ISH of RORbeta and ER81 mRNAs and Fig. 1B shows that of RORbeta and Nurr1 mRNAs in the middle and occipital coronal sections of rat brains. RORbeta and Nurr1 mRNAs showed prominent area differences while ER81 mRNA did not show such conspicuous area difference. As reported previously [14], RORbeta mRNA was most abundant in the barrel field of the parietal cortex area 1 (Par1) (Fig. 1). RORbeta mRNA was generally expressed more abundantly in the sensory areas than in other areas. ER81 mRNA exhibited the opposite pattern, showing higher levels of expression in the areas where the RORbeta mRNA expression level was low (Fig. 1A, see also Fig. 2, panels a, e and f). Nurr1 mRNA exhibited a characteristic area expression pattern (Fig. 1B): its expression in layer 6A was restricted to the lateral regions (e.g., Fig. 1B, par2, Oc2L and Te1, see also Fig. 2, panels b', c', e' and f') and there was only low expression in layer 6B in the dorsal areas (e.g., Fig. 1B, Par1 and Oc1, see also Fig. 2 panels a' and d').

pone-0003266-g001: Double in situ hybridization histochemistry (ISH) of RORbeta/ER81 (A) and RORbeta/Nurr1 (B).Signals in red are for RORbeta and those in green are for ER81 (A) or Nurr1 (B). The arrowheads indicate the area borders that were deduced by comparing the gene expression patterns shown by double ISH and those revealed by the cortical box method. Par1, Par2, Oc1, Oc2L and Te1 correspond to the primary and secondary somatosensory areas (Par1 and Par2), the primary and secondary visual areas (Oc1 and Oc2L) and the primary auditory area (Te1). Ectorhinal cortex (Ect) is also indicated. The white bars denoted as a–e and a'–e' are the regions magnified in Fig. 2. This figure is a montage of several images. Although the lighting condition of the original images was not even at this low resolution, we adjusted the contrast of each component image manually so that the montage appeared to be consecutive. Scale bar, 2 mm.

Mentions:
Figure 1A shows the double ISH of RORbeta and ER81 mRNAs and Fig. 1B shows that of RORbeta and Nurr1 mRNAs in the middle and occipital coronal sections of rat brains. RORbeta and Nurr1 mRNAs showed prominent area differences while ER81 mRNA did not show such conspicuous area difference. As reported previously [14], RORbeta mRNA was most abundant in the barrel field of the parietal cortex area 1 (Par1) (Fig. 1). RORbeta mRNA was generally expressed more abundantly in the sensory areas than in other areas. ER81 mRNA exhibited the opposite pattern, showing higher levels of expression in the areas where the RORbeta mRNA expression level was low (Fig. 1A, see also Fig. 2, panels a, e and f). Nurr1 mRNA exhibited a characteristic area expression pattern (Fig. 1B): its expression in layer 6A was restricted to the lateral regions (e.g., Fig. 1B, par2, Oc2L and Te1, see also Fig. 2, panels b', c', e' and f') and there was only low expression in layer 6B in the dorsal areas (e.g., Fig. 1B, Par1 and Oc1, see also Fig. 2 panels a' and d').

Bottom Line:
Second, at single cell level, RORbeta and ER81 mRNAs were coexpressed in a subpopulation of layer 5 neurons, whereas Nurr1 and ER81 mRNAs were not colocalized.The tight relationship between the expression of the three layer specific genes and functional areas were revealed, demonstrating the usefulness of cortical box method in the study on the cerebral cortex.In particular, it allowed us to perform statistical evaluation and pattern matching, which would become important in interpreting the ever-increasing data of gene expression in the cortex.

Background: The mammalian neocortex is subdivided into many areas, each of which exhibits distinctive lamina architecture. To investigate such area differences in detail, we chose three genes for comparative analyses, namely, RORbeta, ER81 and Nurr1, mRNAs of which have been reported to be mainly expressed in layers 4, 5 and 6, respectively. To analyze their qualitative and quantitative coexpression profiles in the rat neocortex, we used double in situ hybridization (ISH) histochemistry and cortical box method which we previously developed to integrate the data of different staining and individuals in a standard three-dimensional space.

Principal findings: Our new approach resulted in three main observations. First, the three genes showed unique area distribution patterns that are mostly complementary to one another. The patterns revealed by cortical box method matched well with the cytoarchitectonic areas defined by Nissl staining. Second, at single cell level, RORbeta and ER81 mRNAs were coexpressed in a subpopulation of layer 5 neurons, whereas Nurr1 and ER81 mRNAs were not colocalized. Third, principal component analysis showed that the order of hierarchical processing in the cortex correlates well with the expression profiles of these three genes. Based on this analysis, the dysgranular zone (DZ) in the somatosensory area was considered to exhibit a profile of a higher order area, which is consistent with previous proposal.

Conclusions/significance: The tight relationship between the expression of the three layer specific genes and functional areas were revealed, demonstrating the usefulness of cortical box method in the study on the cerebral cortex. In particular, it allowed us to perform statistical evaluation and pattern matching, which would become important in interpreting the ever-increasing data of gene expression in the cortex.